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Diss Kerstin Nagel.Pdf Naturstoffe algenassoziierter Bakterien & Naturstoffe der Gattung Pseudomonas und deren Bedeutung für die Braunalge Saccharina latissima Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Dipl. Ing. (FH) Kerstin Nagel Kiel 2013 II Erster Gutachter: Prof. Dr. Johannes F. Imhoff Zweiter Gutachter: Prof. Dr. Florian Weinberger Tag der mündlichen Prüfung: 18.12.2013 Zum Druck genehmigt: Kiel, 18.12.2013 gez. Prof Dr. Wolfgang J. Duschl, Dekan III Die praktischen Arbeiten wurden am Kieler Wirkstoff-Zentrum am Helm- holtz-Zentrum für Ozeanforschung Kiel GEOMAR an der Christian- Albrechts-Universität zu Kiel von Januar 2008 bis März 2011 unter der An- leitung von Prof. Dr. Johannes F. Imhoff durchgeführt. IV Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit unter Einhaltung der Regeln guter wissenschaftlicher Praxis der Deutschen Forschungsgesell- schaft verfasst habe, und dass sie nach Form und Inhalt meine eigene Ar- beit ist. Außer den angegebenen Quellen und Hilfsmitteln wurden keine weiteren verwendet. Sie wurde keiner anderen Stelle im Rahmen eines Prüfungsverfahrens vorgelegt. Dies ist mein erstes und bisher einziges Promotionsverfahren. (Kerstin Nagel) V Ein Teil der während der Doktorarbeit erzielten Ergebnisse wurde in den folgenden Artikeln bzw. Tagungsbeiträgen veröffentlicht: Publikationen: Wiese, J., Thiel V., Nagel K., Staufenberger T., and Imhoff J. F. (2009). Diversity of antibiotic-active bacteria associated with the brown alga Lam- inaria saccharina from the Baltic Sea. Mar Biotechnol 11:287–300. Nagel, K., Schneemann, I., Kajahn, I., Labes, A., Wiese, J. und Imhoff, J. F. (2012). Beneficial effects of 2,4-diacetylphloroglucinolproducing Pseu- domonads on the marine alga Saccharina latissima. Aquat Microb Ecol, 67 (3). pp. 239-249. Ohlendorf, B., Schulz, D., Erhard, A., Nagel, K. und Imhoff, J. F. (2012). Geranylphenazinediol, an acetylcholinesterase inhibitor produced by a Streptomyces species. J Nat Prod, 75 (7). pp. 1400-1404. Tagungsbeiträge: Wiese, J., Gärtner, A., Heindl, H., Kajahn, I., Krämer, M., Labes, A., Lang, G., Mitova, M., Nagel, K., Schmaljohann, R., Schneemann, I., Stöhr, R., Thiel, V., Yu, Z. und Imhoff, J. F. (2008) Marine microbial resources [Pos- ter] In: 2. Bi-Annual Symposium "The Future Ocean", 06.-09.10, Kiel. Nagel, K., Schneemann, I., Wiese, J., Kajahn, I., Labes, A. und Imhoff, J. F. (2009) Ecological impact of biologically active metabolites produced by Laminaria saccharina associated Pseudomonas sp. strains [Poster] In: 6th European Conference on Marine Natural Products, 19.-23.07, Porto, Por- tugal. Baumann, H. I., Wiese, J., Gärtner, A., Goecke, F. R., Heindl, H., Kajahn, I., Kleinschmidt, K., Labes, A., Nagel, K., Neulinger, S., Ohlendorf, B., Schneemann, I., Schulz, D., Staufenberger, T., Thiel, V., Zinecker, H. und Imhoff, J. F. (2009) Biological role of secondary metabolites from marine microorganisms [Vortrag] In: VAAM-Symposium "Symbiotische Interakti- onen", 19.-20.11, München. Nagel, K., Schneemann, I., Kajahn, I., Wiese, J., Labes, , Lang, G., Goe- cke, F. R., Thiel, V. und Imhoff, J. F. (2009). Ecological impact of biologi- VI cally active metabolites produced by Laminaria saccharina associated Pseudomonas sp. strains [Poster] In: VAAM Jahrestagung, 08.-11.03, Bo- chum. Nagel, K., Schneemann, I., Kajahn, I., Wiese, J., Labes, A. und Imhoff, J. F. (2010) Pseudomonads in association with Saccharina latissima – evi- dence of a beneficial interaction in marine environments? [Poster] In: In- ternational VAAM-Workshop "Biology of bacteria producing natural prod- ucts", 26.-28.09., Tübingen. Labes, A., Nagel, K., Schneemann, I., Wiese, J. und Imhoff, J. F. (2011) From marine ecology to marine biotechnology – Small bioactive molecules in biological interactions and biotechnological applications [Poster] In: Cluster retreat Future Ocean, 28.-29.03., Schleswig. Wiese, J., Schulz, D., Stöhr, R., Nagel, K., Labes, A., Schneemann, I., Heindl, H., Jansen, N., Silber, J., Kramer, A., Goecke, F. R., Schmaljo- hann, R., Staufenberger, T., Gärtner, A., Baumann, H. I., Kleinschmidt, K. und Imhoff, J. F. (2011). Biologically active compounds from microorgan- isms derived from marine macroorganisms and marine sediments [Poster] In: NatPharma: Nature Aided Drug Discovery, NADD, 05.-09.06., Napoli, Italy. INHALTSVERZEICHNIS Inhalt 1 Einleitung ................................................................................... 1 1.1 Entwicklung von Naturstoffen am Beispiel von Antibiotika ..................... 2 1.2 Marine Naturstoffe ........................................................................... 5 1.3 Assoziationen mariner Mikroorganismen ............................................. 8 1.4 Algen ........................................................................................... 10 1.5 Pseudomonaden ............................................................................ 14 1.6 Pflanzen-assoziierte Pseudomonaden ............................................... 15 1.7 Fragestellung dieser Arbeit ............................................................. 17 2 Material und Methoden ................................................................ 19 2.1 Nährmedien und Puffer................................................................... 19 2.2 Chemikalien .................................................................................. 23 2.3 Kits .............................................................................................. 24 2.4 Geräte ......................................................................................... 24 2.5 Probenahme ................................................................................. 26 2.6 Isolierung und Identifizierung der Bakterienstämme .......................... 27 2.7 Konservierung der Bakterienstämme ................................................ 29 2.8 Molekularbiologische Methoden ....................................................... 30 2.8.1 DNA-Extraktion ........................................................................ 30 2.8.2 Polymerase Kettenreaktion ........................................................ 30 2.8.3 Sequenzierung ......................................................................... 31 2.9 Phylogenetischer Stammbaum von Pseudomonas-Stämmen ............... 32 2.10 Untersuchungen zu Pseudomonas sp. LD120 ..................................... 33 2.10.1 BIOLOG GN2 ........................................................................... 33 2.10.2 Substratverwertung .................................................................. 34 2.10.3 Fluoreszensnachweis ................................................................ 34 2.11 Anzucht und Extraktion der Bakterienkulturen ................................... 34 2.11.1 Anzucht auf Agarplatten ............................................................ 34 2.11.2 Anzucht in Flüssigkultur ............................................................ 35 2.11.3 Extraktion der Agarplatten ........................................................ 35 2.11.4 Extraktion der Kulturen im 25/100 mL Maßstab ........................... 35 2.11.5 Extraktion der Kulturen im 1 L Maßstab und Großansatz ............... 36 2.12 Chromatographie ........................................................................... 36 2.12.1 Analytische Chromatographie .................................................... 37 INHALTSVERZEICHNIS 2.12.2 Präparative Chromatographie .................................................... 37 2.12.3 Semi-Präparative Chromatographie ............................................ 38 2.13 Dereplikation ................................................................................ 39 2.14 NMR-Spektroskopie ....................................................................... 39 2.15 Bioassays ..................................................................................... 39 2.15.1 Antibiotische Bioassays ............................................................. 40 2.15.2 Enzyminhibitionsassays ............................................................ 41 2.15.3 Zellkulturbasierte Assays .......................................................... 42 2.15.4 Bakterien aus der KiWiZ-Sammlung ........................................... 43 3 Ergebnisse ................................................................................. 44 3.1 Isolierung biologisch aktiver Bakterien ............................................. 44 3.2 Sind diese Bakterien mit S. latissima assoziiert? ................................ 57 3.3 Physiologisches Profil von Pseudomonas sp. LD120 ............................ 60 3.4 Charakterisierung von Pseudomonas sp. LD120 ................................. 61 3.5 Antimikrobielle Metabolite von Pseudomonas sp. LD120 ..................... 62 3.6 Screening der Naturstoffproduktion Algen-assoziierter Bakterien ......... 62 3.6.1 Naturstoffproduktion von Bacillus sp. (Stamm LB084) .................. 64 3.6.2 Naturstoffproduktion von Kiloniella laminariae (Stamm LD81) ....... 66 3.6.3 Stimulationsexperiment mit Kiloniella laminariae (Stamm LD81) .... 67 3.6.4 Naturstoffproduktion von Pseudoalteromonas sp. (Stamm LB034) .. 69 3.6.5 Naturstoffproduktion von Streptomyces sp. (Stamm LB058) .......... 70 3.6.6 Naturstoffproduktion von Streptomyces sp. (Stamm LB114) .......... 72 3.6.7 Kultivierungsexperimente von Streptomyces sp. (Stamm LB114) ... 74 3.6.8 Naturstoffproduktion von
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